Gene expression signatures of MEF2D-silencing hepatocellular carcinoma cells
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ABSTRACT: Global expression profiles in Huh7 after infection with Lv-shMEF2D and Lv-scrambled, as well as transfection with siRNA-MEF2D and siRNA-control, were compared to investigate the molecular mechanisms involved in MEF2D-mediated regulation of cell cycle. In order to investigate the molecular mechanisms involved in MEF2D-mediated regulation of cell cycle, we assessed gene expression profiles in Huh7 cells after infection with Lv-shMEF2D and Lv-scrambled, as well as transfection with siRNA-MEF2D and siRNA-control, by cDNA microarray. Analysis of global mRNA expression profile indicated a shift toward G2/M arrest in the cells after downregulating MEF2D expression. The genes that inhibit G2/M transition were found to be expressed in high level in MEF2D-downregulated group, as compared with control group. Meanwhile, mRNA abundance of G2/M transition-promoting genes, except CDC2 and CDC25C, was reduced when MEF2D expression was depressed in Huh7 cells
Project description:Global expression profiles in Huh7 after infection with Lv-shMEF2D and Lv-scrambled, as well as transfection with siRNA-MEF2D and siRNA-control, were compared to investigate the molecular mechanisms involved in MEF2D-mediated regulation of cell cycle.
Project description:HBV mRNAs harboring miR-122 response elements were found to bind and sequester endogenous miR-122. This microarray experiment was carried out to find out differentially expressed genes in Huh7 cells expressing HBV mRNAs. RNA was extracted from Huh7 cells transfected with pcDNA3.1 plasmid containing HBV mRNA with wild type (WT) or mutant (Mut) miR-122 response elements in its 3M-bM-^@M-^Y-UTR or the empty pcDNA3.1 vector (3.1) as a mock. Total RNA was then extracted and processed on Affymetrix microarrays in biological duplicate.
Project description:Differentiation and maintenance of cardiac muscle is a complex biological process. MEF2D appears to play an important role in the regulation of cardiomyocyte homeostasis. We knocked down expression of MEF2D in NRVMs, and assessed global expression pattern changes in MEF2D knockdown against a negative control. NRVMs were extracted from 1 to 2 day old rat pups and were allowed to recover for 24 hours before being transduced with shRNA viral vectors to knockdown expression of MEF2D or LacZ (negative control). After 3 days, total RNA was harvested for Affymetrix global gene expression microarray analysis. Each array was pooled RNA from six biological replicates.
Project description:FOXM1 plays a key role in M phase in normal cells and is overexpressed in human glioma. We found that FOXM1 deprivation could sensitize the glioma cells to TMZ chemotherapy. To find out the mechanistic regulation of FOXM1 in chemo-resistant genes, we used microarrays to select the potential genes regulated by FOXM1 which dominates in glioma chemo-resistance. U87 glioma cells were transiently transfected with none-target siRNA or FOXM1 siRNA. Total RNA were extracted after 48 hours and subjected to the microarray.
Project description:Coronary artery disease (CAD) and its complication myocardial infarction (MI) are the leading cause of death worldwide.Our genome-wide association studies (GWAS) in the Chinese population identified a genomic variant, rs6903956, in intron 1 of the C6orf105 gene (later named as ADTRP) as a significant risk factor for CAD and MI. Based on itscell membrane localization and its function on regulation of TFPI, we hypothesize that ADTRP acts as a cell signaling molecule that affects function and expression of many downstream genes/proteins. We performed global gene expression profiling in cells with knockdown of ADTRP expression to identify other downstream targets of ADTRP. To identify other downstream targets of ADTRP, we performed global gene expression profiling in cells with knockdown of ADTRP expression. Because ADTRP downstream genes include those involved in cell cycle regulation and apoptosis as well as multiple histone genes, we carried out cellular studies on cell cycle, cell proliferation and apoptosis to further characterize the function of ADTRP. Global gene expression of ADTRP siRNA sampels and negative controls were profiled by Affymetrix GeneChip PrimeView arrays in HepG2 cells, top downstream genes with differntial expression levels were selected for validation in HepG2, HUVEC, and EAhy926 endothelial cells. Because ADTRP downstream genes include those involved in cell cycle regulation and apoptosis as well as multiple histone genes, we carried out cellular studies on cell cycle, cell proliferation and apoptosis to further characterize the function of ADTRP.
Project description:The objective of our study was to search for survival biomarkers (SB) and treatment response monitoring biomarkers (TRMB) in the urinary proteome of dogs with renal disease secondardy to canine leishmaniosis (CanL),
Project description:Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit extracellular signals from cell surface G protein-coupled receptors to intracellular effector molecules. Attention has been paid to the GM-NM-112 family members because they mediate cell invasion, migration, and tumorigenesis. In our findings, GM-NM-112 levels were higher in human HCCs than non-tumorous liver tissues. Array analyses using Huh7 cells stably transfected with GM-NM-112QL (an active mutant form) enabled us to extract the microRNAs dysregulated by GM-NM-112 active mutant. Of them, miR-122 was most greatly decreased. In addition, we found decreases of miR-200b/a, miR-192/215 levels. Dysregulation of the microRNAs changed the levels of key proteins associated with HCC cell migration/invasion. Huh7 hepatoma cells were transfected with pCMV or the plasmid encoding for activated mutant of GM-NM-112 (GM-NM-112QL). Stable transfectants were selected by incubating the cells in culture medium containing Geneticin for 3 weeks. We performed a miRNA microarray for the identification of global microRNA expression changes using WT-Huh7 and Huh7-GM-NM-112QL cells. RNA for each set was harvested from quadruple plates.
Project description:Microgravity is one of the most important features in spaceflight. Previous evidence has shown that significant changes to the musculoskeletal and immune systems occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human colon colorectal cells that were incubated for 48 or 72 h either in normal conditions and µG simulated conditions. The comparative proteomic method based on the 18O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity.
Project description:Major advances have been made to develop an automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. An in-solution digest strategy is employed to generate peptides from the extracted proteins for LC-MS analysis in the 384-well plate. Method evaluation utilized HeLa cells cultured in the 384-well plate ranging from 500 – 10,000 cells. Digestion efficiency was excellent in comparison to the commercial digest peptides standard with minimal sample loss while improving sample preparation throughput by 20 – 40 fold. Analysis of six human cell types, which included two primary cell samples identified and quantified approximately 4,000 proteins for each sample in a single LC-MS/MS injection with as little as 100 – 10,000 cells depending on cell type demonstrating universality of the platform. Implementation of the comprehensive 384-well format protocol for processing cells to clean digested peptides enables large-scale biomarker validation and compound screening through proteomic analysis.